Purification of pteroic acid amides



Patented Dec. 28, 1948 PURIFICATION OF PTEROIC ACID AMIDES "Brian L.- Hutchings, Montvale, N.. J assignor to I American Cyanamid Company, New, York, N. Y.,

a corporation of Maine No Drawing. Application May 11,1946,

Serial No. 669,099

This invention relates to a process of purifying crude reaction mixtures containing synthetic folic acid andrelated products.

The substance called folic .acid is a growthpromoting material having vitamin-like properties. It'is known to havea stimulating efiect upon the-growth of chicks, rats,.an'd other ani-v mals and upon bacteria, including Streptococcus Zactzs, Lactobacillus casez', and others. product, isolated from natural sources, has been variously designated as folic acid, vitamin Be; factor U, L. casei factor, chick growth factor, and the like. Lately, asubstance'having identical biological activity has been synthesized and shown to be effective in stimulating haemoglobin formation and in the treatment of agranulocytosis, sprue, and other related diseases.

The synthetic production of substances having the identical, or analogous, biological activity of folic acid derived from natural sources has been achieved by reacting 2,4,5-triamino-6-hydroxypyrimidine, an amino acid amide of para-aminobenzoic acid such as para-aminobenzoyl glutamic acid, and an appropriate three carboncompound having reactive aldehyde and/or halogen groups, such as, particularly alpha, beta-dibromopropionaldehyde. L I

Themost important biologically active product of thereaction bears the chemical-name N-[4- (-2 -"amino 4 hydroxy 6 pyrimido[4.5 bl pyrazyl) methyll-amino}benzoyll glutamic acid. Other closely relatedproducts having biologi-' cal activity in varying degree may be prepared by the process by using suitable starting materials in which the glutamic acid residue of the para-'aminoben'zoyl glutamic acid intermediate isre'place'd with another amino acid or apolypeptid thereof, such, for example, being paraaminobenzoylglutamyl glutamylglutamic acid. Several of these synthetic products, particularly the glutamic acid amides of 4-{[(2 -amino4-hydroxy 6 pyrimido [4,5 blpyrazyl) methyl] amino}benzoic acid having one, two, three, and more glutamic acid'radicals connected bya peptide linkage, possess activity similar to folic acid Because ,0f thederived from natural sources. complexity of the nomenclature of these com pounds, the simplified term pteroic acid has,

been suggested and accepted by chemists in the field to designate the fundamental group 4-{[(2- amino 4; hydroxy-6-pyrimido. [4,5-b] pyrazyll The methyl] amino} benzoic acid. For. purposes of, co venience this' simplified term will be used 12 Claims. (Cl. zeta-251).

The chemistry of the glutamic acid amides of pteroic acid are prepared is highlyv complex, particularly inasmuch as the different intermediates mentioned above mayreact in several difierent ways. Chemists familiar with the chemistry of heterocyclic, organiccompounds of this type would expect, and it has been found-by experience, that numerous, side reactions occur along with, or'following, the principal. reaction. Accordingly, the reaction product contains a relatively large proportion of undesired substances, including unreacted intermediates; condensation and/or polymerization products, of'two or more of the intermediates, position isomers, oxidation products, various hypothetical degradation products, products of hydrolysis and the like. Many of these unidentified products'contain a pyrimidopyrazine nucleusv and may be designated generally as pterins.- Some of the by-products of the reaction may be harmful and, inasmuch as the prodnot is tobe used as a therapeutic agent, it is obvious that these pterins should be separated from the biologically active material.

Although folic acid has been isolated from natural sources, with considerable difficulty, however, the processes useful for this purpose have not been found to besuitable for separating synthetic folic acid from the various impurities as sociated with it as a result of the side reactions just mentioned. Unfortunately, many of the pterins associated with-synthetic folic acid in the crude reaction product possess some of the same chemical and physical properties of the desired biologically active product and it has been found extremely di-flicult to separate the impurities without causing an alarming loss of the active material. It is,,therefore, one of the objects of the present invention toprovide a method of purifying; crude reaction mixtures containing glutamic acid amides of pteroic acid to such degreethat the purified product may be used for medical: purposes.

The present invention is based in part upon my discovery that thealkaline earth metal salts of the by-product pterins are more water-insoluble than the alkaline earth-metal salts of the glutamic acid amides of pteroic acid and that this difference is accentuated in alcoholic solution.

In carrying out theprocess of the present invention I prefer to prepare a solution of the crude reaction product at a pH above about 8 using an alkaliflto solubilize the glutamic acid amides of pteroic acid and other pterins in the reaction mixture; The .solution isthen treated with a process by which these acid to such an extent that much of.- it islikewise.

precipitated and lost with the other insoluble pterins.

Ordinarily I prefer to add sufficient-alcohol" to the solution of alkaline earth-metal salts to givethe solution. a *concentratiom-bi frcm about 10%, to20.% byweight of alcohol. when -isolatingl biologically. active: pteroyl' glutamic acid derivatives having: more than one g'Iutamic-acid residue in the molecule; such asN Ei-4-'{ (z-amino- 4'-h.ydroxy-6.-pyrimido ['4,5'-bl i 'pyrazyl) methyl 1- amino}benzoyll glutam ylglutamylglutamic "acid, less alcohol should be used inasmuch 'as' the alkaline earth-metal salts-"of these -polype'ptids' are more insoluble= and excess. alcohol tends to decrease their solubility too much. T ln such cases, therefore, I usefrom -about-=5% to 1'0 of alcohol in :the solution .to depress tna-solubility bathe inactive pterins associated therewith.

-As a result of the selective precipitationbf the unwanted pterins in :the form oiitheir alkaline earthr-metal salts it is possible I to removemost of the impurities with but a nominal loss of the active product. Further purificationm'ay then be'accomplished either by treatment" with activated charcoal :or by extraction of thefreeu-ptero'yl glutamic acidsalts to remove pterinswith other organic solvents. Inasmuch, :howeverp as acti vated charcoal. absorbs thepteroyl giutamic acid almost .as efficientlyastit does the contaminating pterins; fthis technique is n'ot desirable except possibly as a finishing :op'eration." l preferf-there fore, that further purification be' by 'means of organic solvents. v

. After: .most of the contamina'ting pter'ins-lrave been removed by precipitation-- as -allsazline arth metal salts, the product may be-furthebpui ified by butanol extraction. 4 I Kaye/ discovered and this; discovery is also an important'-part the present invention, .that'the contaminating pterinsy which ;are sought to be removed from -'association with :the pteroyl 'glutamic acid-anymore soluble in: butanol than is pteroyl glutamicacid 'The aqueous solution of pteroyl glutam-ic acid-is extractedcwith butanol at a pH between about 6 to 7.5 to remove most of: the remaining pterins' from the partially purified ptero ylglutamic -acid. Several such extractions may barecfuireddoreduce thezcontaminants to thedes-ired-level; -Itr' iS then feasible to treat the aqueous 'solutioir of--' pteroyl glutamic .acid with activated-*charcoai to removethe 'remainingpterins. "Ptero'yl g lutarnic acid may then .be recovered from solution by pre cipitation atpH 3 to'obtain apr'oduct 'of sumcient purity for most medicinal uses.

Example 500 mg. of a crude reactionxprodixctzcontainmg 109.8- mg. of'pteroyl glutamic: acid;the:-remain'der being rpterins andiother .pmdlrctsaofsth' reaction;

is dissolved in 300 ml. of 0.2 N sodium hydroxide solution. Solid barium chloride is then added to 0.2 N. The insoluble precipitate which forms is then removed by filtration and ethyl alcohol is added to the filtrate to a 20% concentration. The precipitate is then removed by filtration and idiscanded..-. p 1 V 'Lf-lhezfiltrate from the. above Asa treated with enough sulfuric acid to precipitate the barium spresent as barium sulfate, an excess of sodium hydroxide being added from time to time, as "necessary; to-keep "the pH of the solution at :..above.8". The filtratezis then diluted to 1200 ml.

andvaddustedtda .pH of about 7 at which more :toi-ithe undesiredmterins precipitate. The solution atthis dilution preferably has a concentra- .;t-i'on,oi 'abo'ut'50 gammas of pteroyl glutamic acid per milliliter. The precipitation of insoluble pterins-at neutrality should be from a solution less concentrated in pteroyl glutamic acid than aboutiflilfi gamm-aapertmil liliter iimthatrthdrmecipitate:tendsrtoecarry downz-withitamuch ofithe. pteroyl glutamic acid when :themteroyl glutamic ac'idnispresent lat. highrconcentrations.

Thecfiltratedatthisstage; of thGglQIQQQSSHQWII tainsi 61.8 :mg. .ofypteroylgglutamic .acid and only 3l.05;-mg-;uof =.the undesired .pterins. Asvwill be "evident; this. represents aziconsiderablerpurifl cationcfthe cnude material.

.i The pterinsnmay iberemoved frontxthe. iiitaiate. above-by treatment with.20.0mg,.vof-. Noriteh. However; the. activated charcoalabsorbs:amapproximatelweqnal;.xamount "oi'ei'the pteizoyli;.'glu.-- tamic; acid and: it:.=is; .prefernedFthat lthismabsora' tionzstepabezomittednrsresenvediorilanai nursificastion; I

The preferred procedure is to extractthe-aque-- ous. solution air-15H 'l -withetemyolurneslofi bntmiol. This.:extraction= ism-repeated; ithree; rtirmescandi re moves .;approximatelys 9il-%l zofiithecnterinsasassociatedrwith the;pteroylsglutamicvacidi. As snrall; amount ofiactiaatedmharcoal;about 1 .gnotieharcoa1.:.io1-: each; Burma;- oinundesirednpterinsgsis. thensadded .tmthe aqueousrsolution and, irheqsclm tiom filtered. "anyxresidualtpigment mesentzmay' bememoxeda with; someclosss biupteroyl; glntamic: acid, by further treatment with charcdala.

.:,Then:aqueous solutiomis themrconcentratedi'in vacuumsozthatr ptemyloglutamic acidsisipresent; at; awconcentrationnof :200. .to- 400 gammas per cubic centimeter. Theci-nsohible; .proiincti itxanyg. at this. 5 stage of .the-= operatiom.is idissolvedlpywarm-ing the solution which islthenl-addustemto 3:,

lated pterins in association therewith wl'iichcomprises the steps of converting the pterins in the mixture to barium salts and separating thewaterinsoluble materials from the water-soluble substances.

3. A process ofseparating glutamic acid amides of 4-{[(2-amino-4-hydroxy-6-pyrimido [4,5-b] pyrazyl) methyl] amino} benzoic acid from related pterins in association therewith which comprises thes'tep of converting the pterins in the mixture to calcium salts and separating the water-insoluble materials from the water-soluble substances.

4.- Aprocess of separating glutamic acid amides of 4-{[(2-aminoi-hydroxy-fi-pyrimido [4,,5-b] pyrazyl 'methyl] amino} benzoic acid fromrelated pterins in association therewith which comprises tlie step of converting the pterins in the mixtur'e'to alkaline earth-metal salts and subjecting the'mixed salts to the action of an aqueous solution containing not more than about 25% of a lower aliphatic alcohol and separating lthe water-insoluble materials from the water-soluble substances.

5. A process of separating N-[-4-{[(2-amino-4- hydroxy-B-pyrimido [4,5-b] pyrazyl) methyl] amino} benzoyl] glutamic acid having the formula:

00011 N t N OHRNHOOONH H H I (SH: N N N 1H:

OOH

from related pterins in association therewith which comprises the step of converting the pterins in the mixture to barium salts and subjecting the mixed salts to the action of an aqueous solution containing not more than about 25% of ethyl alcohol and separating the water-insoluble materials from the water-soluble substances.

6. A process of separating N- [-4-{[ (2-amino-4- hydroxy-G-pyrimido [4,5-b] pyrazyl) methyl] amino} benzoyl] glutamic acid having the formula:

OH COOH N t N OHiNHOCONH H NH I OH: N N 43H;

from related pterins in association therewith which comprises the step of converting the pterins in the mixture to alkaline earth-metal salts, subjecting the mixed salts to the action of an aqueous solution containing not more than about 25% of a water miscible alcohol and separating the insoluble materials from the soluble substances, eliminating alkaline earth-metal from the solution and thereafter extracting the water-soluble aqueous fraction with butanol to remove butanolsoluble products.

7. A process of separating N- [-4-{[(2-amino-4- hydroxy-G-pyrimido [4,5-b] pyrazyl) methyl] amino} benzoyl] glutamic acid having the formula:

I 00011 N t N CHaNH 0 ONE H NH (5H: NAN Hi OOH N 5 N CHaNHOCONH H T a N N.

H: (EH1 A $0011 from related pterins in association therewith which comprises the step of treating an aqueous solution of the mixture with a water-soluble calcium compound to convert the soluble pterins to calcium salts and separating from the soluble fraction those products insoluble in an aqueous solution containing from 5 to 20% by weight of ethyl alcohol.

9. A process of separating N-[4-{[(2-amino-4- hydroxy-G-pyrimido [4,5-b] pyrazyl) methyl] amino} benzoyl] glutamylglutamylglutamic acid having the formula CHQNH CONH H Cfiz COOH NH a (5 N N 0N}! H H: COOH ONH H OOH from related pterins in association therewith which comprises the step of treating an aqueous solution of the mixture with a water-soluble barium compound to convert the soluble pterins to barium salts and separating from the soluble fraction those products insoluble in an aqueous solution containing not more than about 10% of ethyl alcohol.

10. A process of separating N-[4-{[(2-amino- 4-hydroxy6-pyrimido [4,5-b] pyrazyl) methyl] amino} benzoyl] glutamic acid from related pterins in association therewith which comprises the step of treating an aqueous solution of the mixture with a water-soluble barium compound to convert the soluble pterins to barium salts and separating from the soluble fraction those products insoluble in an aqueous solution containing from 5 to 20% by weight of a water miscible alcohol, eliminating barium from the solution by precipitation as barium sulfate, adjusting the hydrogen ion concentration of the solution to about pH 7 and separating insoluble pterins therefrom, extracting butanol-soluble products from the solution by extraction with butanol and thereafter adjusting the hydrogen ion concentration of the aqueous phase to about pH 3 to obtain a precipitate of N- [4{[(2-amino-4-hydroxy-6-pymamas rimido [4;5-b] pyrazyl) methyl] amino} benzoyl] glutamic acid. 1 v 11. A process of separating N- E4-{It2-am'ino- 4hydroxy-6--pyrimido [4,5-b] pyrazyll' methyl] amino} benzoylil' glutamic acid from related pterin's in association therewith "which: comprises theustep of treating an aqueous solution of the mixture with a water-soluble calcium compound to convert the soluble pterins to calcium salts and separating from the soluble fraction 'those'produc-ts insoluble in an aqueous solution containing from 5 to 20% by weight of a water miscible alcohol, eliminating calcium from the solution by precipitation as calcium sulfate, adjusting the hydro-.

gen ion concentration of the solution to about pH '7 and separating insoluble pterins therefrom, extracting butanol-soluble products from the solution by extraction with'butanol and thereafter adjusting the hydrogen ion concentration of, the aqueous phase to about pH 3 to obtain "a precipitate of N-[4-{[(2-amino-4-hydroxy-G-pyrimido [4,5- b] pyrazyl) methyl] amino} benzoyl] glutamic acid. 1 r 12. process of separating N-lf-4-'{.[(2-arxiino- 4-hydroxy-6-pyrimido [4,540] pyrazyll methyl] ill amino} benzoyl] glutamylglutamic acid having the formula:

a, coon cosmos can No references cited. 

